Determination of ochratoxin A in beer marketed in Spain by liquid chromatography with fluorescence detection using lead hydroxyacetate as a clean-up agent

A new sample treatment for liquid chromatographic analysis of ochratoxin A (OTA) in beer is proposed. Degassed beer is mixed with lead hydroxyacetate, which precipitates some bulk components but does not remove OTA. The precipitate is separated and the acidified liquid is extracted with chloroform. The solvent is evaporated and the residue is dissolved in mobile phase (acetonitrile-water, 40:60, v/v; acidified at pH 3.0 with phosphoric acid) and separated by liquid chromatography using fluorescence detection. The limit of detection was 0.005 ng/ml. The average recovery rate and the average RSD of recovery in the spiking level range 0.01-0.5 ng/ml were 95.5% and about 5%, respectively. The method is cheaper that other alternative ones using immunoaffinity columns or other solid-phase extraction cleanup:The separation was optimised with regard to composition and flow of the mobile phase and no interference from the matrix was found. The method was applied to 88 samples of beer (domestic and imported) marketed in Spain. OTA was detected in 82.9% of them. The range for positive samples was 0.007-0.204 ng of OTA/ml.     

RP-HPLC analysis of phenolic compounds and flavonoids in beverages and plant extracts using a CoulArray detector

Methods were developed for the analysis of natural antioxidants including phenolic compounds and flavonoids in beverages and plant extracts using gradient HPLC with multi-channel electrochemical coulometric detection. Suitability of various reversed-phase columns for this purpose was compared; pH and mobile phase gradients were optimized with respect to the separation selectivity and sensitivity of detection. Because of different target compounds in various sample types, the overlapping resolution maps and the normalized resolution product approaches described earlier were used to select optimum columns and gradients to suit the analysis of the individual sample types. The methods were applied to the analysis of phenolic compounds and flavonoids in beer, wine, tea, and yacon extracts. 32 phenolic compounds were identified and determined, including derivatives of benzoic and cinnamic acids, flavones, and a few related glycosides. Eight-channel CoulArray detection offers high selectivity and sensitivity with limits of detection in the low microg L(-1) range, at least an order of magnitude lower than single-channel coulometric detection using the Coulochem detector. No special sample pretreatment is necessary and, because of the compatibility of the CoulArray detector with gradient elution, phenolic antioxidants of different polarities can be determined in a single run. In addition to the retention times, the ratios of the areas of the pre-dominant and post-dominant peaks to the area of the dominant peak can be used for improved identification of natural antioxidants.     

Dual gradient LC method for the determination of pharmaceutical residues in environmental samples using a monolithic silica reversed phase column

Detailed below is a simple reversed-phase liquid chromatography (RP-LC) method for the simultaneous separation of up to 21 acidic, basic, and neutral pharmaceuticals using Merck Chromolith Performance RP-C18e monolithic columns with direct ultraviolet (UV) absorption detection. By simultaneously applying a solvent elution gradient program with a mobile phase flow gradient, both a decrease in the overall analysis time and a general increase in peak efficiencies were observed. Mobile phase pH and buffer concentration were optimised using the overall resolution product under applied gradient conditions. Under optimised conditions peak area reproducibility (n?=?6) ranged between 0.4 and 9.3%, determined at the method LOQ level. For real sample analysis pharmaceutical residues were extracted using an optimised solid phase extraction (SPE) procedure, utilising Strata-X extraction cartridges, which overall provided the highest relative recovery data in comparison with four other commercially available SPE sorbents (17 out of 20 residues investigated had recoveries over 70%). Complete method precision, including all sample pre-treatment and LC analysis for six spiked river water samples at the 1 and 2?µg?L^?1 level was between 10 and 29%. Using 1?L volumes of 1?µg?L^?1 spiked estuarine water samples, the majority of detection limits were found to be in the 10–50?ng?L^?1 range.     

Electrochemical behaviour of sertraline at a hanging mercury drop electrode and its determination in pharmaceutical products

The electrochemical behaviour of sertraline at a hanging mercury drop electrode (HMDE) was described. Different voltammetric techniques, such as cyclic, linear sweep, differential pulse and square wave voltammetry, were used. Voltammograms were obtained at different pH values with a Britton-Robinson buffer solution used as supporting electrolyte. The best results were found by square wave voltammetry with electrodeposition at alkaline pH using a borate buffer with a pH = 8.2 for the samples, containing 12% (v/v) methanol. Under optimised conditions, a linear relationship between 2.33 x 10(-7) and 3.15 x 10(-6) M of sertraline with a limit of detection of 1.98 x 10(-7) M was obtained. The electrochemical method developed was applied to the determination of sertraline in pharmaceutical formulations. Recoveries were close to 100%, thus proving efficacy of the proposed method for the quantification of sertraline in commercial samples.     

High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aqueous ionic eluents. II. Application of UV, fluorescence and electrochemical oxidation detection

Unmodified silica columns together with non-aqueous ionic eluents give stable yet flexible systems for the analysis of basic drugs by high-performance liquid chromatography. Low-wavelength UV and fluorescence detection may be used, and fluorescence may be optimised by, for example, post-column pH change or derivatisation of some primary aliphatic amines with o-phthaldialdehyde. A novel feature is that electrochemical oxidation can be used for the detection of most analytes and this detection mode is thus discussed in detail. Retention and relative response data (UV, 254 nm and electrochemical, +1.2 V) have been generated for 462 compounds using a 125-mm Spherisorb S5W silica column and methanolic ammonium perchlorate (10 mM, pH 6.7) as eluent. This system can be used isocratically in qualitative analyses and also for quantitative work, when either the wavelength or the applied potential can be adjusted to optimise the response.     

Electrochemical behaviour of sertraline at a hanging mercury drop electrode and its determination in pharmaceutical products

The electrochemical behaviour of sertraline at a hanging mercury drop electrode (HMDE) was described. Different voltammetric techniques, such as cyclic, linear sweep, differential pulse and square wave voltammetry, were used. Voltammograms were obtained at different pH values with a Britton-Robinson buffer solution used as supporting electrolyte. The best results were found by square wave voltammetry with electrodeposition at alkaline pH using a borate buffer with a pH = 8.2 for the samples, containing 12% (v/v) methanol. Under optimised conditions, a linear relationship between 2.33 × 10^–7 and 3.15 × 10^–6 M of sertraline with a limit of detection of 1.98 × 10^–7 M was obtained. The electrochemical method developed was applied to the determination of sertraline in pharmaceutical formulations. Recoveries were close to 100%, thus proving efficacy of the proposed method for the quantification of sertraline in commercial samples. Received: 26 September 2000 / Revised: 1 December 2000 / Accepted: 5 December 2000     

Magnetic mixed hemimicelles solid-phase extraction of xanthohumol in beer coupled with high-performance liquid chromatography determination

In this study, silica-coated magnetic nanoparticles (Fe(3)O(4)/SiO(2) NPs) modified by cetyltrimethylammonium bromide (CTAB) were synthesized. They were successfully applied for extraction of xanthohumol in beer based on magnetic mixed hemimicelles solid-phase extraction (MMHSPE) coupled with high-performance liquid chromatography-ultraviolet determination. The main factors influencing the extraction efficiency including the surfactant amount, the beer pH, the extraction time, the desorption condition and the maximum extraction beer volume were optimized. Under the optimized conditions, a concentration factor of 60 was achieved by extracting 120 mL beer sample using MMHSPE and the detection limit of xanthohumol is 0.0006 mg/L. The proposed method was successfully applied for determination of xanthohumol in various beer samples with the xanthohumol contents in the range of 0.031-0.567 mg/L. The satisfactory recoveries (90-103%) were obtained in analyzing spiked beer samples.     

High-efficiency peptide analysis on monolithic multimode capillary columns: Pressure-assisted capillary electrochromatography/capillary electrophoresis coupled to UV and electrospray ionization-mass spectrometry

High-efficiency peptide analysis using multimode pressure-assisted capillary electrochromatography/capillary electrophoresis (pCEC/pCE) monolithic polymeric columns and the separation of model peptide mixtures and protein digests by isocratic and gradient elution under an applied electric field with UV and electrospray ionization-mass spectrometry (ESI-MS) detection is demonstrated. Capillary multipurpose columns were prepared in silanized fused-silica capillaries of 50, 75, and 100 microm inner diameters by thermally induced in situ copolymerization of methacrylic monomers in the presence of n-propanol and formamide as porogens and azobisisobutyronitrile as initiator. N-Ethylbutylamine was used to modify the chromatographic surface of the monolith from neutral to cationic. Monolithic columns were termed as multipurpose or multimode columns because they showed mixed modes of separation mechanisms under different conditions. Anion-exchange separation ability in the liquid chromatography (LC) mode can be determined by the cationic chromatographic surface of the monolith. At acidic pH and high voltage across the column, the monolithic stationary phase provided conditions for predominantly capillary electrophoretic migration of peptides. At basic pH and electric field across the column, enhanced chromatographic retention of peptides on monolithic capillary column made CEC mechanisms of migration responsible for separation. The role of pressure, ionic strength, pH, and organic content of the mobile phase on chromatographic performance was investigated. High efficiencies (exceeding 300 000 plates/m) of the monolithic columns for peptide separations are shown using volatile and nonvolatile, acidic and basic buffers. Good reproducibility and robustness of isocratic and gradient elution pressure-assisted CEC/CE separations were achieved for both UV and ESI-MS detection. Manipulation of the electric field and gradient conditions allowed high-throughput analysis of complex peptide mixtures. A simple design of sheathless electrospray emitter provided effective and robust low dead volume interfacing of monolithic multimode columns with ESI-MS. Gradient elution pressure-assisted mixed-mode separation CE/CEC-ESI-MS mass fingerprinting and data-dependent pCE/pCEC-ESI-MS/MS analysis of a bovine serum albumin (BSA) tryptic digest in less than 5 min yielding high sequence coverage (73%) demonstrated the potential of the method.     

Determination of direct violet 51 dye in water based on its decolorisation by electrochemical treatment

The decolorisation process of a synthetic textile dye, direct violet 51, was investigated in an aqueous solution using an electrochemical method in a batch electrochemical cell. Graphite electrodes were used as the anode and cathode for the decolorisation process. The parameters such as applied current, initial pH, solution conductivity, interfering ions, and effect of electrodes were optimised. It was found that the dye with an initial concentration of 20 mg L^?1 could be removed after 50 min using a current of 100 mA with colour removal of up to 94 %. The UV-VIS spectra of the dye were analysed prior to and after treatment and these confirmed that the conjugated systems were decomposed at a current of 100 mA. The optimised method was successfully applied to real wastewater samples.     

Fabrication of a nanostructured TiO2/carbon nanotube composite electrode for voltammetric and impedimetric determination of NTO explosive in the water and soil samples

An electrochemical oxidation route was developed for sensitive and selective assay of nitrotriazolone (NTO) explosive in some environmental samples on a multi-walled carbon nanotube (MWCNTs)/TiO₂ nanocomposite paste electrode, for prevention of the analytical interference of conventional reducible energetic compounds. Detailed evaluations were made for the electrochemical behaviour of NTO on the modified electrode by adsorptive stripping voltammetry, electrochemical impedance spectroscopy (EIS) and chronoamperometry techniques in the pH range of 2.0–10.0. Parameters such as diffusion coefficient constant of NTO were calculated, and various experimental conditions were also optimised. Under optimal conditions the calibration curve had two linear dynamic ranges of 130.0–3251.5 μg L^?1 and 6.5–26.0 mg L^?1 with a detection limit of 26.0 μg L^?1 (0.2 μmol L^?1) and precision of <3%. This electrochemical sensor was further applied to determine NTO in real soil and water samples with satisfactory results.     

Investigating the effects of protein patterns on microorganism identification by high-performance liquid chromatography-mass spectrometry and protein database searches

A method based on liquid chromatography coupled to mass spectrometry with positive electrospray ionisation was developed for the analysis of cyanobacterial hepatotoxins in environmental samples. The chromatographic separation was performed using two microbore columns, 2 mm and 1 mm I.D. columns, which allowed the coupling of liquid chromatography to mass spectrometry with no flow splitting. Analytes were eluted using two different water-acetonitrile, both acidified with formic acid gradients. Mass spectrometric parameters were optimised in order to maximise sensitivity. Detection limits for the 2 mm I.D. column ranged from 0.077 to 2.057 ng in full scan and from 0.021 to 1.153 ng in SIM mode. However, limits of detection as low as 60-340 pg in full scan and 6-72 pg in SIM mode were achieved for the 1 mm I.D. column. Finally, the proposed method was applied to the analysis of microcystins and nodularins in real samples.     

Preconcentration and dansylation of aliphatic amines using C18 solid-phase packings. Application to the screening analysis in environmental water samples

Precolumn preconcentration and derivatization on solid sorbents (Bond Elut C18 solid-phase extraction cartridges) of low-molecular-mass aliphatic amines in water samples have been performed using dansyl chloride (Dns-Cl) as derivatization reagent. Conditions for analyte preconcentration and derivatization such as volume sample, reagent concentration, time, pH and temperature reaction were optimised. On the basis of these studies a rapid and sensitive method for screening of aliphatic amines in waters is presented. Up to volumes of 5 ml, samples are drawn through the sorbent, the analytes retained are dansylated at basic pH, at 100 degrees C for 10 min or 85 degrees C for 15 min. The derivatized analytes are desorbed with 0.5 ml of acetonitrile. Twenty microl of the collected extracts are chromatographed in a Hypersyl ODS C18 column using an acetonitrile-imidazole (pH 7) gradient for elution. Seven amines and ammonium were separated within 9 min. The Dns derivatives were monitored at 333 nm with UV detection and at lambda(excitation) = 350 nm and lambda(emission) = 530 nm with fluorescence detection. The different signals are compared. Dynamic ranges from 10 to 250 microg/l and limits of detection at the microgram-per-litre level and relative standard deviations from 2 to 15% were obtained for all the amines. The total analysis time (sample treatment plus chromatography) was less than 25 min. The method was applied to determination and screening analysis of these analytes in real environmental water samples.     

A pre-concentration procedure employing a new imprinted polymer for the determination of copper in water

This work describes the development by response surface methodology (RSM) of a procedure for copper determination by inductively coupled plasma optical emission spectrometry (ICP OES) in water samples after extraction by copper imprinted polymer. Results of the two-level full factorial design (2⁴) based on an analysis of variance demonstrated that only the solution pH; amount of polymer and adsorption time were statistically significant. Optimal conditions for the extraction of copper samples were obtained by using Box-Behnken design. Solution pH; amount of polymer and adsorption time were regarded as factors in the optimisation study. The working conditions were 4.6, 0.03?g and 3.5?h, for solution pH, amount of polymer; and adsorption time, respectively. Under the optimised experimental conditions, the detection limit of the proposed method followed by ICP OES was found to be 0.8?µg?L^?1. The relative standard deviation (RSD) was found to less than 0.81%. The pre-concentration factor was 22.5. The accuracy of the optimised procedure was evaluated by analysis of certified reference material. The method was applied to the determination of copper in water samples.     

Determination of phenolic compounds and hydroxymethylfurfural in meads using high performance liquid chromatography with coulometric-array and UV detection

The objective of this study was the determination of 25 phenolic compounds in different mead samples (honeywines) using high performance liquid chromatography (HPLC) with coulometric-array detection and in case of hydroxymethylfurfural with UV detection. Our method was optimized in respect to both the separation selectivity of individual phenolic compounds and the maximum sensitivity with the electrochemical detection. The method development included the optimization of mobile phase composition, the pH value, conditions of the gradient elution and the flow rate using a window-diagram approach. The developed method was used for the determination of limits of detection and limits of quantitation for individual compounds. The linearity of calibration curves, accuracy and precision (intra- and inter-day) at three concentration levels (low, middle and high concentration range) were verified. Mead samples were diluted with the mobile phase at 1:1 to 1:50 ratio depending on the concentration and filtered through a PTFE filter without any other sample pre-treatment. Phenolic compounds concentration was determined in 50 real samples of meads and correlated with meads composition and hydroxymethylfurfural concentration. The most frequently occurred compounds were protocatechuic acid and vanillic acid (both of them were present in 98% samples), the least occurred compounds were (+)-catechin (10% samples) and sinapic acid (12% samples). Vanillin and ethylvanillin, which are used as artificial additives for the taste improvement, were found in 60% and 42% samples, respectively. Hydroxymethylfurfural concentration, as an indicator of honey quality, was in the range from 2.47 to 158 mg/L. Our method is applicable for the determination of 25 phenolic compounds in mead, honey and related natural samples.     

Simultaneous determination of multiple constituents in real beer samples of different origins by capillary zone electrophoresis

Simultaneous determination of alcohols, amines, amino acids, flavonoids, and purine and pyrimidine bases in bottled beer samples directly without any pre-treatment was carried out by capillary zone electrophoresis with diode-array detection. Electrolyte conditions such as pH, composition and concentration of the buffer, working voltage and type and time of injection were checked. The best separation of the cited analytes was achieved in 70 mM sodium borate solution and pH 10.25. The detection limits were from 2.1 to 5.6 mg L^–1 for the 18 compounds studied. The developed method is rapid, sensitive and quantitative and has been applied to seven types of international bottled beers of different origins bought locally.     

Routine determination of urinary free catecholamines by high-performance liquid chromatography with electrochemical detection

A simple and reliable high-performance liquid chromatographic method is described for the routine determination of the free catecholamines (norepinephrine, epinephrine and dopamine) in urine. The catecholamines are isolated from urine samples using small affinity chromatography columns prepacked with immobilised m-aminophenylboronic acid, separated by ion-pair reversed-phase liquid chromatography and quantified by electrochemical detection. Total analysis, including sample preparation time, is achieved in less than 30 min with analytical recoveries of 92-96% for all three catecholamines. Long-term stability and reproducibility of the liquid chromatographic system is attained by selection of optimised conditions for chromatographic separation with a formate mobile phase and produces detection limits of 1.4, 1.8 and 2.2 nmol/l for norepinephrine, epinephrine and dopamine, respectively, in urine samples and day-to-day coefficients of variation of less than 6%. Furthermore, the affinity isolation gels can be reused a minimum of ten times providing a rapid and cost-effective means of sample preparation.     

Isocratic Liquid Chromatography with Segmented Columns and Simultaneous UV and Dual Electrochemical Detection: Application to the Selectivity Enhancement for the Determination of Explosives

The concept of stationary‐phase optimized liquid chromatography (POPLC) was studied in the context of separation and detection aspects for the determination of explosives. A tailor‐made segmented column assembly with three complementary reverse‐phase materials enabled the separation of 14 priority target nitroaromatic explosives. This experimental approach resulted in excellent selectivity without using gradient‐elution protocols which can considerably deteriorate the performance of electrochemical detection. Consequently, the POPLC separations could be operated under isocratic conditions which were very important for the reliable use of electrochemical detection. Simultaneous UV and dual electrochemical detection resulting in three detection traces was exploited as an additional means for the further enhancement of selectivity in more complex samples.     

High-performance liquid chromatographic separation and quantification of citric, lactic, malic, oxalic and tartaric acids using a post-column photochemical reaction and chemiluminescence detection

An HPLC method was developed for the determination of citric, lactic, malic, oxalic and tartaric acids by chemiluminescent detection following online irradiation with visible light. The organic acids were irradiated with visible light in the presence of Fe3+ and UO2(2+) to generate Fe2+, which was determined by measuring the chemiluminescence intensity in a luminol system in the absence of added oxidant. Factors affecting the photochemical and chemiluminescence reactions were optimised so that their contribution to the total band-broadening was negligible. The chromatographic separation was performed on a C18 column under isocratic reversed-phase conditions using 0.005 M H2SO4 mobile phase. The optimised method was validated with respect to linearity, precision, limits of detection and quantification, accuracy specificity and robustness. The applicability of the assay was demonstrated by analysing these compounds in real samples such as milk, fruit juices, soft drinks, wine and beer.     

Highly Sensitive Choline Oxidase Enzyme Inhibition Biosensor for Lead Ions Based on Multiwalled Carbon Nanotube Modified Glassy Carbon Electrodes

The determination of lead ions by inhibition of choline oxidase enzyme has been evaluated for the first time using an amperometric choline biosensor. Choline oxidase (ChOx) was immobilized on a glassy carbon electrode (GCE) modified with multiwalled carbon nanotubes (MWCNT) through cross‐linking with glutaraldehyde. In the presence of ChOx, choline was enzymatically oxidized into betaine at –0.3 V versus Ag/AgCl reference electrode, lead ion inhibition of enzyme activity causing a decrease in the choline oxidation current. The experimental conditions were optimised regarding applied potential, buffer pH, enzyme and substrate concentration and incubation time. Under the best conditions for measurement of the lowest concentrations of lead ions, the ChOx/MWCNT/GCE gave a linear response from 0.1 to 1.0 nM Pb²⁺ and a detection limit of 0.04 nM. The inhibition of ChOx by lead ions was also studied by electrochemical impedance spectroscopy, but had a narrower linear response range and low sensitivity. The inhibition biosensor exhibited high selectivity towards lead ions and was successfully applied to their determination in tap water samples.     

Column-switching high-performance liquid chromatographic detection of pholcodine and its metabolites in urine with fluorescence and electrochemical detection.

A sensitive and selective method for the detection of pholcodine and its metabolite morphine in urine using high-performance liquid chromatography is described. It involves on-line clean-up of urine on a trace enrichment column packed with a polymeric strong cation-exchange material. Pholcodine and its metabolites were separated on two analytical columns with different selectivities. Pholcodine was detected by a fluorescence detector and morphine was detected electrochemically. One system, based on reversed-phase chromatography, applied a polystyrene-divinylbenzene column and gradient elution. The other system was based on normal-phase chromatography with a silica column and isocratic elution. Morphine was confirmed to be a metabolite of pholcodine by reversed-phase chromatography and electrochemical detection. Two unidentified metabolites of pholcodine were separated from pholcodine by normal-phase chromatography and detected by fluorescence detection.